Characteristics of CclSAUR49 expression and its effect on limonoids biosynthesis in citrus

Abstract:【Objective】Limonoids are secondary metabolites widely distributed in the Rutaceae and Meliaceae, with biological and pharmacological activities such as anti-oxidation, anti-allergy, anti-obesity, anti-osteoporosis, anti-cancer, anti-bacteria, anti-virus. The genetic modulation of limonoids biosynthe- sis is still poorly understood. So the investigation of genes regulating the limonoids biosynthesis and the study of their functions will help to understand the regulatory mechanism of limonoids biosynthesis and provide a theoretical basis for further cultivation of high-quality citrus. It has been shown that the SAURsignificantly affects the biological yield and production of secondary metabolites by regulating the hor- mone metabolism in the plant, but whether it is involved in the biosynthesis of limonoids has not been reported. To study the role of CclSAUR49 gene in biosynthesis of limonoids and characteristics of its promoter in citrus, the open reading frame (ORF) and promoter of CclSAUR49 gene were cloned from citrus. Limonoids contents, gene expression levels, subcellular location and promoter activity were ana- lyzed.【Methods】Limonoids contents and expression of the CclSAUR49 in the leaves of different devel- opmental stages from two genotypes of Shatianyou pomelos [Citrus grandis (L.) Osbeck] varied in li- monoids content were analyzed with HPLC and Real-time qRT-PCR. Specific primers were designed to amplify the target fragment using the sequence in the Clementine genome (http: //phytozome.jgi.doe.gov/pz/portal.) as a reference. The coding region and promoter of the CclSAUR49 were isolated from Shatianyou pomelo and precocious trifoliate orange (Poncirus trifoliata L. Raf.) via PCR amplification, respectively. Subcellular localization of the CclSAUR49 protein was accomplished with transient ex- pression of 35S::SAUR49-GFP fusion protein in the leaves of Nicotiana benthamiana and observed us- ing confocal microscope. cis-acting element of the promoter was predicted through the PlantCARE web- site. In order to analyze the characteristics of the promoter, pSAUR49::GUS fusion expression vector was constructed and transformed into precocious trifoliate orange. Using uninfested precocious trifoli- ate orange as a control, the whole positive plants were stained chemically with GUS (beta-glucuroni- dase) and decoloured with 75% ethanol until the chlorophyll in the leaves of the plants had completely faded to a white colour and then observed and photographed.The overexpression vector of the pSAUR49::GFP fusion protein was constructed and transformed into precocious trifoliate orange. Resis- tant shoots were obtained and grafted on potted rootstock. Limonoids content and expression of the Ccl- SAUR49 gene in transgenic plants were analyzed, the length of leaf and internode and circumference of stem were determined three months after grafting.【Results】HPLC results showed that the limonoids content of the two Shatianyou genotypes was significantly different, with STY2 containing significantly more limonoids than STY1. The contents of nomilin and limonin were continuously decreasing along with the growth and development of leaves, while the gene expression level of the CclSAUR49 showed an increasing trend. This revealed that limonoids contents and levels of the CclSAUR49 expression dem- onstrated a significant negative correlation during the development of the leaf with correlation coeffi- cients of -0.978, -0.958 (p<0.01) in STY1 and STY2, respectively. Subcellular localization analysis in- dicated that CclSUAR49 protein was located in the plasma membrane and nucleus. A 1600 bp fragment upstream of the CclSAUR49 gene start codon (ATG) was cloned from citrus. The results of bioinformat- ic analysis predicted several types of cis-acting elements in the promoter of the CclSUAR49 gene, but no AuxREs -responsive elements had been found. Six transgenic seedlings of the CclSUAR49 and GUSfusion expression were obtained by citrus genetic transformation. The results of GUS staining transgen- ic seedlings indicated that GUS was mainly expressed in the stems, roots and old leaves, but not in the young leaves. Two transgenic seedlings of the CclSUAR49 and GFP fusion expression were obtained. Comparing the growth performance of the positive transgenic with the control plants grown under the same conditions, significant morphological difference was observed in the transgenic plants with larger size of leaves, longer internodes and thicker stems. Compared with the wild type control, both nomilin and limonin content were significantly reduced, with reductions of 44.81% and 22.60% of nomilin con- tent and 23.74% and 67.36% of limonin content in OE-1 and OE-2, respectively. qRT-PCR results showed that the expression levels of the CclSAUR49 was largely and significantly increased in the over- expression transgenic plants, with 661 and 156 fold increases in OE-1 and OE-2, respectively. Analysis of the expression of the CclSAUR49 in relation to the limonoids content revealed a significant negative correlation with correlation coefficients of-0.983 (p<0.01) and-0.975 (p<0.01) in OE-1 and OE-2, respectively. In order to clarify the role of the CclSAUR49 gene in the limonoids biosyntheses, the ex- pression analysis was also performed on the six genes associated with limonoids synthesis identified by previous transcriptomic analysis. The overexpression of the CclSAUR49 affected the expression of those genes, suggesting that the CclSAUR49 might interact with some of those genes to regulate the bio- synthesis or accumulation of limonoids in citrus.【Conclusion】The CclSAUR49 gene was a tissue-spe- cific expression gene. It negatively affected the biosynthesis or accumulation of limonoids in citrus.